3,5,3′-triiodothyronine sulfate as thyromimetic agent and pharmaceutical formulations thereof

ABSTRACT

The invention regards the use of triiodothyronine sulfate, commonly named T 3 S, as a medicament having thyromimetic activity for the treatment of pathologies due to organic deficiency of triiodothyronine (T 3 ), as such or in association with thyroxine (T 4 ), and pharmaceutical formulations for oral administration thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of and claims priority to U.S. Ser. No.13/083,047, filed Apr. 8, 2011, now published as US-2011-0245342-A1 onOct. 6, 2011, and subsequently abandoned as of Oct. 22, 2013, all ofwhich are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention regards the use of 3,5,3′-triiodothyroninesulfate, usually named triiodothyronine sulfate or T₃ sulfate (T₃S), asan active principle, alone or in combination with thyroxine, in thetreatment of pathologies due to organic deficiency of3,5,3′-triiodothyronine. Accordingly, the same is usable for thepreparation of thyromimetic pharmaceutical compositions.

BACKGROUND OF THE INVENTION

A number of iodothyronines are present in blood, which are directlyproduced by thyroid gland or are the result of peripheral metabolism ofother iodothyronines. Among them, 3,5,3′-triiodothyronine (acronym T₃)is deemed to be the biological active form of thyroid hormone (TH),because it has shown high affinity for the specific receptors of thesame and is normally present in serum at a concentration sufficient forthe activation of said receptors.

The main secretion product of thyroid gland in the healthy adult isthyroxine, commonly designated with the acronym T₄. It is periphericallyconverted to its biologically active form, T₃ (Ref.1), through enzymaticremoval of an iodine atom from the external aromatic ring of themolecule by both type I and type II 5′-iodothyronine monodeiodinases(type I MD and type II MD, respectively). This metabolic pathway is themain mechanism of endogenous production of T₃; thus, T₄ can properly beconsidered a pro-hormone. On the other hand, a minor part of T₃ is alsodirectly secreted by thyroid. On average, the amount of T₄ produced inan adult being of 70 Kg weight every day amounts to 100 μg, while thetotal production of T₃ amounts to around 25 μg. 4-8 μg of T₃ out of said25 μg are directly secreted by thyroid and the remaining ones derivefrom the peripheral conversion of T₄.

T₃ undergoes two different metabolic pathways. The main metabolicpathway consists in the partial deiodination of the inner aromatic ringby type III 5-iodothyronine monodeiodinase (type III MD) to give3,3′-diiodothyronine, which is biologically non-active and is furthermetabolized through deiodination or sulfoconjugation. The othermetabolic pathway regards around 20% of the total amount of T₃ producedby the body and brings on sulfoconjugation of T₃ to give T₃S, which isnot able to bond to the thyroid hormones (Ref.2), thus resultingbiologically non-active (Ref.3).

Contrary to what happens with T₃, T₃S is not deiodinated by type III MD.Rather, it is an excellent substrate for type I MD (Ref.4), whichconverts it very quickly into 3,3′-diiodothyronine sulphate. Thus it hasbeen widespread common knowledge that, in the healthy adult being,sulfoconjugation of T₃ to give T₃S represents a way for speeding up thecatabolism of T₃, so facilitating its biliary and urinary excretion.Actually, it was found that serum levels of T₃S, physiologically low inthe healthy adult, are higher when type I MD activity is reduced.

Yet, it was found that, just in some body districts and organs,sulfatases exist which, under particular physiological conditions andsituations, are able to convert again T₃S into its active form T₃(Ref's.7-9).

Such enzymes have been described in the intestinal microflora as well asin body tissues like liver, kidneys and nervous central system (Ref.10).

Recently, it has been found that endogenous T₃S levels in serum arequite high during intrauterine life and as such are kept by the body,i.e. higher than the ones normally found in the adult being, at leastuntil the forth month of postnatal life (Ref.11). Considering theessential role played by thyroid hormones during growth, in particularas far as nervous central system functions are involved, hypotheses havebeen made about the possibility that, in this tissue, T₃S may alsopossibly be used by the body as an occasional source of T₃, if and whenneeded, during the first period of life. Studies performed on autopticspecimens of human nervous cerebral tissue post-mortem showed that theamount of T₃ in the same results limited by type III MD (Ref.12). Whilethis enzyme does not attack T₃S, it has been surmised that T₃S mayexceptionally represent an alternative endogenous source of T₃ hormonein those tissues which contain sulfatases able to reconvert T₃S into itsactive form, just in case a particular need of the hormone arises insaid tissues (Ref's.8, 13).

Further studies have been performed, aimed at ascertaining the effectiverole played by T₃S during production and metabolism of thyroid hormones.Said studies have recently demonstrated that when administered byintraperitoneal (i.p) administration in single or 3 to 10 daily doses athyromimetic effect is observed in hypothyroid rats (Ref.10). Ineuthyroid rats (Ref.14) T3S, administered i.p., shows a thyromimeticeffect on several parameters such as body weight and TSH serum levels.In both references T₃S has shown a potency of around one fifth that ofT₃. Moreover both treatments with T₃S and with T₃ produced a significantreduction of serum levels of thyreotropic hormone (TSH) in euthyroidrats, thus showing to possess similar capability in inhibiting itssecretion. On the contrary, in the case of hypothyroid rats, T₃S showeda poor capability of inhibiting TSH secretion when compared to T₃. It iswell known that TSH is a highly responsive indicator to the functionalstatus of thyroid gland and detects the smallest alterations of itshormonal secretion. Actually, its levels are higher under conditions ofreduced thyroid functionality, even in those conditions that are definedas sub-clinical, while they are reduced when an excess of thyroidhormones are present. As a consequence, T₃S activity seemsnon-comparable to T₃ as far as its capability of inhibition on formationof TSH is involved.

Therefore, particularly in view of the latest studies the biologicalrole of T₃S is still controversial.

In fact, its main, well-grounded and universally accepted, feature isits non-biological activity, i.e. it is a biologically inert metaboliteof T₃ (Ref's.2 and 3), and the sulfation pathway is regarded as ametabolic activator of T₃ catabolism (Ref.5).

On the other hand, only in particular tissues and under exceptionalcritical conditions due to shortage of thyroid hormone in those tissues,it has been shown its potential as an endogenous local source of T₃.

As a result, today the skilled technician is still facing a complex,somewhat conflicting, situation, which highlights only some of thebiological characteristics of the product and needs more exhaustive indepth studies.

To the best of our knowledge, however, none of the several documentsforming the state-of-the-art discloses, shows or suggests thepossibility of using this metabolite of T₃ in therapy. No closeprior-art document, either of experimental nature or substantiallyspeculative, either taken alone or in combination with other relateddocuments, suggests the use, or even the potential use of T₃S as amedicament, taken as such or preferably in combination with otherthyroid hormones or pro-hormones, like, for example T₄. The fact that,only in some specific tissues of the body and under particular, peculiarcircumstances, part of T₃S can be reconverted into T₃ does not mean, norimplies, nor suggests that it is possible to generalize this feature tothe whole organism through exogenous administration of the product. Inparticular, there is no suggestion that oral administration of theproduct, even in protected form according to known methods of thepharmaceutical technique, may render it bioavailable also because it iswell known that in those districts where suitable sulfatases are notpresent the same is rapidly metabolized and excreted through the bileand urines.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Panel a) T₃S calibration curve by competitive ELISA; Panel b)DELFIA calibration curve. T₃S was assayed at 33.6, 56, 93.3, 155.5, 259,432, 720, 1200, 2000 pg/mL.

FIG. 2. Schematic of DTPA-T₃S monoamide synthesis.

FIG. 3. Mean serum concentrations of T₃S (ng/dl) levels in the fourdoses groups (native values).

FIG. 4: Mean serum concentration curves of T₃S and Total T₃ afteradministration at a dose of 160 μg.

SUMMARY OF THE INVENTION

It has now unexpectedly been found, and this is one of the aspects ofthe present invention, that T₃S (triiodothyronine or3,5-diiodo-O-[3-iodo-4-(sulphoxy)phenyl]-L-thyronine), as the onlyactive principle in a suitable composition or in association with otherthyroid hormones or pro-hormones, preferably T₄ (Tyroxine, or3,5,3′,5′-tetraiodothyronine) and properly formulated according to thedesired application for oral administration is particularly useful as amedicament to be used in all those pathologies caused by insufficientproduction by the body of the needed quantities of active thyroidhormones, in particular T₃.

Another aspect of the present invention is a non-radioactive immunoassayfor T₃S quantitation, preferably based on chemiluminescence and thereagents developed therein.

A further object of the present invention is a method for thetherapeutic treatment of a hypothyroid condition, which comprises theoral administration of T₃S or the combination T₃S and T₄ as a thyroidhormone replacement therapy to a subject in need thereof.

According to a preferred embodiment oral administration is accomplishedby solid compositions, preferably in the tablet form, comprising T₃Salone or in combination with a second active principle, tyroxine (T₄),in a dose comprised for T₃S of from 1 to 1000 μg, preferably 2.5-500 μgand T₄ of from 1 to 800 μg.

Preferred active principle quantities in the formulation comprising twoactive principles, are the following: T₃S 5-250 μg and T₄ 5-400 μg, T₃S10-100 μg and T₄ 10-200 μg.

In any case, the preferred ratio between active principles (T₄:T₃S) iscomprised from 10:1 to 0.1:1, with a more preferred range comprised from5:1 to 1:1. Even more preferred is the range comprised from 3:1 to 2:1(T₄:T₃S).

Said compositions include diluents, glidants or lubricants anddisintegrants and in a preferred embodiment consist essentially of:calcium carbonate, glycerol dibehenate, croscarmellose sodium salt,hydrate colloidal silica, magnesium stearate and microcrystallinecellulose, in an even more preferred embodiment these components arepresent in the amounts described below for a 80-150 mg tablet:

Calcium carbonate 20-40 mg, preferably 25-35 mg, more preferably 30 mgGlycerol dibehenate 2-15 mg, preferably 4-8 mg, more preferably 5 mgCroscarmellose sodium salt 1-10 mg, preferably 2-6 mg, more preferably3.5 mg Hydrate colloidal silica 0.1-5 mg, preferably 0.5-4, morepreferably 2 mg Magnesium stearate 0.01-2 mg, preferably 0.1-1 mg, morepreferably 0.5 mg Microcrystalline cellulose Up to 110 mg

This composition is endowed with optimal dissolution rates and stabilityof the active principle(s) for at least 24 months.

DETAILED DESCRIPTION OF THE INVENTION

It has been now been surprisingly found that T₃S or salts thereof is notonly a physiologic T₃ catabolite, but that it may be also provided as adrug and administered in hypothyroid conditions. Accordingly, thisrepresents the main aspect of the invention.

Furthermore, it has been found that T₃S, once administered is convertedto T₃ and allows maintenance of steady levels of T₃ in the body for longtimes (from 12 to at least 18 hrs, more preferably at least 48 hrs).This is not only particularly useful when a supplement thyroid hormonein its most active form is needed, but, once more, is completelyunexpected given the rapid metabolism synthetic T₃ undergoes onceadministered. In this case in fact a peak level is detected in serum atabout 2-3 hours and after that rapidly and completely cleared from bloodwithin 12-24 hrs.

Furthermore, it has also been found that T₃S can be administered orallyand this represents a further and particularly advantageous aspect ofthe invention. In fact its biological activity, measured for example bytotal T₃ levels in the serum of thyrectomized individuals, is detectedafter oral administration. This result is quite unexpected because, atvariance with thyroid hormones which are not very soluble in water, T₃Sor salts thereof is a polar molecule whose gastrointestinal absorptionwas expected to be rather inefficient. Therefore the present inventiondiscloses that oral administration is possible and that by this route:a) T₃S is found in the bloodstream thus demonstrating that it crossesthe gastrointestinal barrier, b) is converted into the more activethyroid hormone, T₃, c) maintains T₃ levels in serum afteradministration for quite a long time (at least 48 hours).

According to this finding, a further object of the present invention isrepresented by a method for the therapeutic treatment of a hypothyroidcondition, which comprises the oral administration of T₃S or thecombination T₃S and T₄ as a thyroid hormone replacement therapy for asubject in need thereof. Clinical signs of hypothyroidism are thefollowing: asthenia, fatigue, skin dryness, somnolence, speech fluencyimpairment, cold intolerance, weight gain and/or memory deficit.Accordingly, any of these conditions may be improved by the oraladministration of T₃S alone or in combination with T₄.

In general, oral administration of T₃S and salts thereof inpharmaceutical compositions is proposed according to a preferred aspectof the invention for treating any hypothyiroid conditions or for any T₃replacement therapy. The therapeutic treatment comprises administeringcompositions comprising T₃S, either alone in a dose comprised for T₃S offrom 1 to 1000 μg, preferably from 2.5 to 500 μg, more preferably from 5to 250 μg, or in combination with a second active principle, thyroxine(T₄) (combination compositions) wherein T₄ is present from 1 to 800 μg.

Preferred active principle quantities in the combination compositionsare the following: T₃S 5-250 μg and T₄ 5-400 μg, T₃S 10-100 μg and T₄10-200 μg.

In any case, for combination compositions a preferred ratio betweenactive principles (T₄:T₃S) is comprised from 10:1 to 0.1:1, with a morepreferred range comprised from 5:1 to 1:1. Even more preferred is therange comprised from 3:1 to 2:1 (T4:T₃S).

Preferred administration of the compositions is in a single daily dosageform.

Particularly preferred in the therapy of hypothyroidism, representing afurther aspect of the present invention, is the association of T₃S withT₄. The hormonal association which, in theory, should more accuratelymime the normal thyroid secretion is represented by a combination of T₄with T₃. Actually, pharmaceutical compositions comprising both of saidiodothyronines, formulated in proportions similar to the ones of thenormal physiologic secretion, have already been tried and marketed.Unfortunately, the oral simultaneous administration of T₄ with T₃ wasnot able to reproduce the normal thyroid hormones serum levels, becauseof pharmacokinetics of T₃. In fact, T₃ undergoes a very quick absorptionand an equally quick elimination after oral administration; itselimination rate is about 20 times higher than the one of T₄. For thisreason administration of T₃ gives raise to a dangerous peak excess inhormone concentration, if compared to the normal physiologic levels,followed by a much too fast drop to sub-physiologic levels. Thus, todaymost of the specialised physicians prefer using T₄ alone, even if inthis way production of T₃ only depends on the periferic deiodination ofT₄, because direct secretion of T₃ by thyroid does not exists or isseriously insufficient.

On the contrary, the association of the invention avoids the aboveproblems, because it has unexpectedly been found that, for example,after oral administration, T₃S provides T₃ serum levels that increase ina gradual way and keep steady for long periods of time, thus preventingthe formation of too high peaks.

Another unexpected advantage deriving from the use of T₃S in thetreatment of pathologies due to organic deficiency of T₃ consists in itsrecently found systemic thyromimetic activity linked to a poorinhibition of TSH secretion. This effect is particularly useful in thecase of thyroidectomized patients suffering from thyroid carcinoma, whenadministration of T₄ must be suspended in view of carrying outradiotherapy. In such a case administration of T₃S instead of T₄ mayalleviate a patient's symptoms or disease, such as asthenia, fatigue,skin dryness, somnolence, speech fluency impairment, cold intolerance,weight gain and/or memory deficit without interfering with radioactiveiodine (usually ¹³¹I) radiotherapy.

According to this observation, a further aspect of the invention relatesto T₃S administration in thyroidectomized patients in case of ¹³¹Iradiotherapy when T₄ administration must be suspended. In fact, T4 isusually suspended at least 40 days before radiotherapy to allow anoptimal radioisotope uptake. The lack of thyroid hormones for such along time is usually very badly tolerated by the organism which iscompletely depleted of thyroid hormones within a few days, thussuffering from asthenia, fatigue, skin dryness, somnolence, speechfluency impairment, cold intolerance, weight gain or memory deficit. Incontrast, T₃S, due to its low thyromimetic properties, can beadministered, preferably by the oral route, up to at least 5 days, morepreferably up to at least 4, 3, or 2 days, before radiotherapy.

Another further advantage of T₃S in the therapy of hypothyroidismregards its autolimitation capability. In fact, it is activelydeiodinated by type I MD, which, on its part, is stimulated by thyroidhormones. In hypothyroid subjects type I MD activity is reduced; thusT₃S elimination is slowed. As a matter of fact, its effect on the bodyis greater. On the contrary, in case of over administration, type I MDactivity is increased, thus giving more T₃S elimination, i.e. limitingpossible undesired collateral effects.

Last but not least, a further advantage of T₃S is represented by thefact that it is a metabolite normally present in the body, usuallynon-active, i.e. non-toxic.

Accordingly, another main aspect of the present invention regardspharmaceutical formulations comprising T₃S as an active principle, assuch or in combination with other thyroid hormones or pro-hormones.Particularly preferred are formulations comprising T₃S in associationwith T₄.

The preferred ratio of the two active principles (T₄:T₃S) in compositioncomprising both active principles ranges from 10:1 to 0.1:1, with a morepreferred range comprised from 5:1 to 1:1. Even more preferred is therange comprised from 3:1 to 2:1 (T₄:T₃S).

Said formulations differ in the dosage of the active principle orprinciples, or in the type of pharmaceutical form provided, depending onthe administration route used with enteral administration beingpreferred.

According to this embodiment, compositions for oral administration,either liquid or solid are both suitable. Preferred liquid compositionsshould take into account the generally poor solubility of thyroidhormones such as T₄ and salts thereof, as well as the usually goodsolubility of T₃ sulphate and salts thereof. Furthermore, the use oflactose, glucose and sucrose should be avoided.

The preparation of specific pharmaceutical formulations in response toparticular needs will be described in the following.

Solid Compositions.

It is known that thyroid hormones and especially levothyroxine sodiumare compatible with some excipients but incompatible with others.Carbohydrates, such as starch and maltodextrin, are compatible withthyroid hormones, whereas lactose, glucose and sucrose, have beendetermined to be incompatible. By the use of suitable compatiblediluents, glidants or lubricants and disintegrants, thyroid hormones canbe formulated into tablets, capsules, or powder dosage forms.

Thus, preferred compositions of the present invention are prepared inthe substantial absence of lactose, glucose, sucrose,polyvinylpyrrolidone, and/or a Poloxamer. According to this embodiment,the solid composition comprises diluents or fillers, glidants and/orlubricants and disintegrants. The compositions may also further compriseexcipients, stabilizers, preservatives or dissolution enhancers.

Preferred diluents are cellulose derivatives, such as microcrystallinecellulose, powdered cellulose, silicified microcrystalline cellulose,cellulose acetate, ethyl- or methylcellulose or salts thereof. However,other diluents may be used, such as kaolin, starch and derivativesthereof, or sodium or other alkaline inorganic salts such as trisodiumphosphate, tricalcium phosphate, calcium carbonate or magnesiumcarbonate.

Suitable disintegrants for use in the present invention include cornstarch, croscarmellose and salts thereof (i.e. croscarmellose sodium)and crospovidone or salts thereof. However other disintegrants may beused such as, polymethacrylates and maltodextrin or salts thereof,pregelatinized starch and sodium starch glycolate or sodium alginate,sometimes referred to as diluents as well.

Suitable lubricants for use in the present invention comprise silicatesin general, including colloidal silicon dioxide hydrate silicon dioxide,hydrate colloidal silica, talc, as well as magnesium or zinc stearatethe preferred ones.

Suitable glidants or lubricants are chosen among glycerol dibehenate,tribasic calcium phosphate, starch derivatives, talc, magnesium and zincstearate, sodium stearate fumarate and sodium and magnesium laurylsulphate. Preferred glidants or lubricants are glycerol dibehenate ortribasic calcium phosphate.

The term glidant comprises agents working also as lubricants, and forthose, such as talc, magnesium or zinc stearate or sodium dibehenate,accordingly, classification might be interchangeable.

Flavorants and colorants may be added if desired as additional optionalingredients.

Thyroid hormones, especially levothyroxine sodium and T₃S areparticularly stable in connection with cellulose derivatives. Accordingto this embodiment solid dosage compositions with improved and superior,stability, content uniformity, good tableting and dissolution propertieswhich comprise T₃S or salts thereof alone or in combination with T₄ orsalts thereof in the quantities above disclosed further in combinationwith a cellulose derivative, wherein microcrystalline cellulose orsilicified microcrystalline cellulose are particularly preferred.

Thyroid hormones are preferably prepared by the synthetic route (e.g. asdescribed for T₃S in Mol and Visser, Endocrinology 1985, 117 N. 1,1:1-8).

In the solid composition, diluents are preferably present in apredominant amount, preferably in the range of 50 to 99.99% by weight.More preferably they are present in an amount of from 60 to 80% byweight, more preferably from 65-75% by weight.

According to a preferred embodiment, cellulose or derivatives thereofare present and preferably a second diluent is also present, preferablycalcium carbonate, up to 35% of the total diluent w/w.

Preferred glidants, are selected from the group consisting of glyceroldibehnate (most preferred), talc and silica derivatives, among whichmagnesium trisilicate, starch or derivatives thereof, amides, tribasiccalcium phosphate, are usually present in the composition in a quantityrange from 1 to 10%, most preferably 4 to 6% (w/w).

Lubricants are preferably selected in the group consisting of: magnesiumor zinc stearate, hydrate colloidal silica and talc, more preferablymagnesium stearate and hydrate colloidal silica, in a total quantity offrom 0.1 to 7% even more preferably the first one comprised from 0.1 to2% and the second comprised from 0.5 to 5%.

Disintegrants for use in the present invention include starch,croscarmellose sodium and crospovidone. Preferred is croscarmellose orsodium salts thereof in a quantity ranging from 0.5 to 10% even morepreferably comprised from 1-5%, most preferably comprised from 2- to 4%.

The moisture content of the solid dosage form, such as of a capsule ortablet, is also important. It is preferred that the moisture content isnot higher than 15%, even more preferably not higher than 10%. A buffersystem may be present as a stabilizer in the solid dosage form.

A significant advantage of the preparations of the present invention isthat they can be prepared as a direct compression formula, drygranulation formula, or as a wet granulation formula, with or withoutpreblending of the drug, although preferably with preblending, and stillachieve remarkable stability of the resulting solid dosage formpreparation.

The amount of the thyroid hormone in the preparations of the presentinvention can vary widely, mainly depending on the administrationprotocol. However, due to the high potency exhibited by most of thethyroid hormones, and especially levothyroxine sodium, normally very lowamounts of this thyroid hormone will be utilized.

The solid compositions comprising T₃S alone comprise T₃S of from 1 to1000 μg; according to a further embodiment they comprises also T₄(tyroxine): according to this embodiment (combination compositions) T₃Sis present in a quantity of from 2.5-500 μg and T₄ of from 1 to 800 μg.

Even more preferred active principle quantities in the formulationcomprising two active principles are the following: T₃S 5-250 μg and T₄5-400 μg, T₃S 10-100 μg and T₄ 10-200 μg.

In any case, the preferred ratio between active principles (T₄:T₃S) iscomprised from 10:1 to 0.1:1, with a more preferred range comprised from5:1 to 1:1. Even more preferred is the range comprised from 3:1 to 2:1(T₄:T₃S).

In the present specification, solid composition percent values refers toweight/weight (w/w) ratios and the pharmaceutical dosage form is ofabout 50-200 mg.

The compositions of the present invention are usually prepared byblending the thyroid hormones with microcrystalline cellulose, calciumcarbonate, glycerol dibehenate, crosscarmellose salt, hydrate colloidalsilica.

The resulting blend can be lubricated with magnesium stearate andtableted using a tablet press.

According to the invention, the solid composition of the invention areprepared in tablets and comprise either T₃S as the only active principleor T₃S in combination with a second active principle, preferably T₄, inthe quantities and ratios above indicated, together with the followingdiluents, disintegrants, glidants, lubricants or excipients. In apreferred embodiment, the composition contains 1 to 1000 μg T₃S, in amore preferred embodiment the compositions include 2.5 to 500 μg T₃S ormore preferably 5-250 μg T₃S and the following further ingredients (inamounts described below for a 80-150 mg tablet):

Amount per Tablet Calcium carbonate 20-40 mg, preferably 25-35 mg, morepreferably 30 mg Glycerol dibehenate 2-15 mg, preferably 4-8 mg, morepreferably 5 mg Croscarmellose sodium salt 1-10 mg, preferably 2-6 mg,more preferably 3.5 mg Hydrate colloidal silica 0.1-5 mg, preferably0.5-4, more preferably 2 mg Magnesium stearate 0.01-2 mg, preferably0.1-1 mg, more preferably 0.5 mg Microcrystalline cellulose Up to 110 mg

For combination compositions T₃S is preferably present in a quantity offrom 2.5-500 μg and T₄ of from 1 to 800 μg, or, even more preferably:T₃S: 5-250 μg and T₄: 5-400 μg, or T₃S: 10-100 μg and T₄ 10-200 μg.

It is intended that the above quantities preferably refer to about 110mg tablets, preferably for daily single dosage administration, eventhough the skilled artisan may envisage adjustments due to alternativecomposition forms and/or therapeutic treatment protocols. Due to theconversion rates of T₃S to T₃ within the body a single administrationevery two or three days may be also envisaged.

According to the embodiment above for tablets, the following compositionfor T₃S or T₄ and T₃S as active principle(s) (0.01-1% w/w) represents afurther object of the present invention:

-   -   a diluent selected from cellulose or derivatives thereof,        preferably together with a second diluent, preferably calcium        carbonate, up to 35% of the total diluent (w/w);    -   a glidant, selected from glycerol dibehnate (most preferred),        talc, silica derivatives among which magnesium trisilicate,        amides, tribasic calcium phosphate, are usually present in the        composition in a quantity range from 1 to 10%, most preferably 4        to 6% (w/w);    -   a disintegrant selected from starch, croscarmellose sodium and        crospovidone. Preferred is croscarmellose sodium salt in a        quantity ranging from 0.5 to 10% even more preferably comprised        from 1-5%, most preferably comprised from 2- to 4% (w/w);    -   a lubricant selected from magnesium stearate, hydrate colloidal        silica and talc, more preferably magnesium stearate and        colloidal silica, in a total quantity range comprised from 0.1        to 7% even more preferably the first one comprised from 0.1 to        2% and the second comprised from 0.5 to 5% (w/w).

Different tablet weight and active principle contents or differentadministration protocols may be envisaged for those skilled in the art.

The tablets according to the preferred embodiment show optimaldissolution rates (see table below) and an optimal stability of theactive principle(s) (at least 24 months).

The following properties measured in conditions according to ICHGuidelines:

Dissolution test ≧75% after 45′ Moisture content   ≦10% Resistance tocrushing ≧20N HPLC Title T3S 90-110% HPLC Title T4 90-110%

Liquid compositions, for example obtainable by crushing one or moretablets and dissolving such a mixture or the blended mixture in aqueoussolutions are also possible. Optionally, trace amount (i.e. below 5%) ofa pharmaceutically acceptable antioxidant is also present. It iscontemplated that such compounds may include, for example, ammoniumchloride and/or one or more iodide donors (e.g., sodium iodide).

The compositions of this invention may further comprise one or morephysiologically acceptable formulation excipients, such as thosedescribed in “Remington, J. P, Gennaro, A. R., Remington'sPharmaceutical Sciences, Mack Publishers, Easton, (20th Edition, 2000).

The compositions of this invention are particularly suitable for oraladministration.

The term “oral formulation” means that the active ingredient(s) isformulated into a product suitable for administering to an animal viathe mouth. These formulations may include apart from the solidcompositions described above, for example, liquids or semi-liquids,gels, pastes, oral sprays, buccal formulations, or animal feedscontaining the active ingredients. Said liquid or semi-liquidcompositions are typically aqueous solution.

The pharmaceutical compositions described above are prepared preferablyas tablets, obtainable by direct compression of the mixture abovedescribed in powder. In some such embodiments, for example, the processfurther comprises combining T₃S or T₃S and T₄ in the solid compositionsdescribed above with an aqueous composition optionally comprising abuffer for the preparation of a pharmaceutical liquid composition fororal administration.

It should be kept in mind that when the association is taken intoaccount, the formulations of the present invention will also possiblycomprise individually formulated doses of T₃S and T₄, for sequential orcombined administration. In this case, one suitable kit is provided,which permits administration of said active principles in ways that candiffer from patient to patient, depending on the needed therapeuticapplication. In such a way, the specialized physician will have a widechoice of changing the prescription according to the actual need of thepatient.

Just by way of a non-limitative example, in the case of oraladministration, one package containing two individual blisters, whichhave different shape and/or color and/or different contents and/ordoses, may suit the desired scope. Other possibilities exist and areeasily available to the expert of the field.

The pharmaceutical compositions of the present invention are usable inthe treatment of pathologies due to organic deficiency oftriiodothyronine (T₃), like, for example, original hypothyroidism fromautoimmune thyroid affections, hormonal production defects,thyroidectomy, congenital hypothyroidism, as well as some disorders dueto reduced activity of type I 5′-iodothyronine monodeiodinase (type IMD) which is induced, for example, by hypothyroidism, non thyroidalsystemic illnesses, fast, selenium shortage and so on.

Once administered to a human patient in need thereof, preferablysuffering from a hypothyroid conditions, T₃S levels can be measured andadjusted accordingly by a non-radioactive method immunoassay, eitherbased on colorimetric, fluorescent or chemiluminescent detection.

Accordingly, a further embodiment of the invention is represented by anon radioactive immunoassay. Preferably the immunoassay is an EnzymeLinked Immuno Assay (ELISA), more preferably a competitive ELISA inwhich increasing amounts of T₃S compete for the binding to a solid phasebound anti-T₃S antibody, (e.g. the polyclonal disclosed in Chopra etal., J. Clin. Endocrinol. Metab., 1992, 75: 189-194) with a fixed amountof T₃S conjugated with an avidin-derivative detectable moiety (i.e.biotin), preferably carried out in a multi-well plate. More preferably,the avidin-derivative is a streptavidin and the detectable moietycomprises a chemiluminescent moiety (such as Alkaline Phosphatase orHorseradish Peroxidase), preferably HRP. The use of biotin-avidininteraction, combined with the various detection luminescence astechniques for signal development, allows signal amplification andincreased sensitivity, comparable to a RIA test (see i.e. Chopra et al.,J. Clin. Endocrinol. Metab., 1992, 75: 189-194) but without the need forradioactivity—a clear advantage over the prior art. The ELISA assay, theT₃S-biotin reagent and its synthesis, and kits for T₃S quantitationcomprising such reagent, represent a further object of the presentinvention.

As an alternative embodiment, the T₃S immunoassay is developed for aparticular fluorescence technique, known as DELFIA® (DissociationEnhanced Lanthanide to Fluorescence ImmunoAssay) by which the requiredsensititvity is obtained. This assay, the synthesized reagents, and kitsfor T₃S quantitation comprising said reagents represent a further objectof the invention.

Thus, accordingly, a DTPA-T₃S monoamide(3,5-Diiodo-N-[[(carboxymethyl)[2-[(carboxymethyl)[2-[bis(carboxymethyl)amino]ethyl]amino]ethyl]amino]acetyl]-O-[3-iodo-4-(sulfooxy)phenyl]-L-tyrosine)of Formula I, represents a chelating compound according to a preferredembodiment:

Other molecules can be designed and synthesized by an expert in thefield, through conjugation of T₃S with a variety of chelating moieties,among those suitable for complexation of lanthanide ions, e.g.,nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA),ethylenediamine-N,N′-bis(2-hydroxyphenylacetic acid) (EDDHA),ethylenediaminedisuccinic acid (EDDS), propanediaminetetraacetic acid(PDTA), diethylenetriaminetetraacetic acid (DTTA),diethylenetriaminepentaacetic acid (DTPA), and similar molecules.Conjugation between the chelating agent and T₃S can be obtained by avariety of methods already known to the expert in the field, including adirect amide bond formation, as exemplified in Experimental Part, or theuse of bifunctional chelating agents, that may even be commercialproducts, such as(S)-1-p-isothiocyanatobenzyldiethylenetriaminepentaacetic acid (DTPAisothiocyanate—Invitrogen cat. I24221), or similar products.

Suitable lanthanide metals to be used as chelate labels are selected inthe group consisting of: samarium, terbium, dysprosium and europium.

Particularly preferred is the Europium chelate3,5-Diiodo-N-[[(carboxymethy)[2-[(carboxymethyl)[2-[bis(carboxymethyl)amino]ethyl]amino]ethyl]amino]acetyl]-O-[3-iodo-4-(sulfooxy)phenyl]-L-tyrosine(Formula II).

A schematic of its synthesis is shown in FIG. 2 and can be summarized asfollows: DTPA dianhydride is partially hydrolysed by adding anapproximately equimolar amount of water dissolved in a suitable organicsolvent, then the product, mainly composed of DTPA monoanhydride isreacted with T₃S, in presence of a suitable organic or inorganic base.After solvent evaporation, the oily residue is diluted with water. Theresulting precipitate is collected, washed with water and dissolved inan water/acetone mixture. This crude reaction product is purified on acolumn of Amberlite XAD 1600, or similar resin, developing with mixturesor gradients of water/acetone. The product containing fractions arecollected and evaporated to dryness, yielding the desired DTPA-T₃Smonoamide.

Lanthanide complexation is obtained according to known procedures byadding an equimolar amount of a lanthanide salt to the monoamide watersolution and adjusting the pH at 7 with a suitable base (e.g. NaOH).Optionally, the lanthanide chelated product can be desalted byadsorption on a resin column (e.g. Amberlite XAD1600) and elution withwater/solvent mixtures.

Also in this case, a sensitivity comparable to the RIA test (see Chopraet al., ibidem) is obtained and the use of radioactive isotopes avoidedthis represents a clear advantage over the prior art.

According to a further embodiment, the invention comprises a kit for T₃Sadministration and dosage in serum, wherein said kit comprises anadministration/therapeutic kit with a number of T₃S or T₃S and T₄composition daily doses (i.e. the weekly, bi-weekly, monthly orbi-monthly need), preferably in the form of tablets as described above,and a dosage kit for T₃S immunodetection by a non-radioactive assay.

A further preferred embodiment of the kit comprises tablets with bothactive principles and a kit for T₃S immunodetection in serum. In apreferred embodiment the immunodetection is an ELISA test as describedabove.

The kit comprises a container for a non radioactive immunoassayaccording to the alternative embodiments described above and containerfor the weekly need of the solid daily dosage described above. Thecontainer for the solid daily dosage may be formulated for the weekly,bimonthly, monthly or even multiple months needs, according to thepatients and therapeutic treatment need.

EXPERIMENTAL SECTION

As an example, absolutely non-limiting for the skilled technician, T₃Smay be administered for oral use at doses ranging from 1 to 1000 μg,preferably from 2.5 to 500 μg, more preferably from 5 to 250 μg.

Analogously, when in association with T₄, preferred doses range from2.5-500 μg of T₃S and 1 to 800 T₄ or, even more preferably: T₃S: 5-250μg and T₄: 5-400 μg, or T₃S: 10-100 μg and T₄ 10-200 μg.

Two representative formulations for oral administration, selected amongthe preferred ones, are hereinafter enclosed by way of an example.Obviously, said formulations have no limiting effect on the otherpossible variations, which may also comprise different types ofadministration, different doses or different components depending on thespecific pharmacological application or the particular pathology.

Example A Oral Formulation Containing T₃S

T₃S 50 μg; Calcium phosphate dibasic anhydrous 103.5 mg; Mais starch17.65 mg; Microcrystalline cellulose 5 mg; Sodium carboxymethylamide 5mg; Talc 5 mg; Citric acid 2.8 mg; Magnesium stearate 1 mg

Example B Oral Formulation Containing T₃S and T₄

T₃S 50 μg; T₄ sodium salt 125 μg; Calcium phosphate dibasic anhydrous103.5 mg; Mais starch 17.525 mg; Microcrystalline cellulose 5 mg; Sodiumcarboxymethylamide 5 mg; Talc 5 mg; Citric acid 2.8 mg; Magnesiumstearate 1 mg

Example C Tablets Comprising T₃S

Pre Mixture

In a 2-L amber glass of mixer Turbola transfer a portion Avicel PH102and a T₃S salt and mix for 5′±15″.

Final Mixture

In a stainless steel tank of double cone mixer, transfer the pre-mixturea second portion of Avicel PH102. Mix for 10 minutes at 10 RPM.

The mix was sieved in 1-mm opening sieve and the remaining excipients:Compritol 888 ATO, Syloid 244, Acdisol, Magnesium stearate and Socal S2VDC and the last portion of Avicel PH102 were added directly in thedouble cone mixer, with mixing for 20 minutes at 10 rpm.

The mixture was pressed in 110-mg tablets by a rotary tabletting machineequipped with 7-mm diameter, round, flat jewls, with one-sidedbreak-mark in the middle.

Tablets were shown to have chemical and physical characteristicsaccording to ICH Guidelines.

Tablets were prepared and have shown to have the followingcharacteristics:

20 μg dosage (25° C./60%)

Dissolution test ≧75% after 45′ t₀ = 104.8%; t_(3 mesi) = 103.5%;t_(6 mesi) = 103% Moisture content   ≦10% Resistence to crushing ≧20NHPLC Title T3S 90-110% HPLC Title T4 90-110%100 μg dosage (25° C./60%)

Dissolution test ≧75% after 45′ t₀ = 94.4%; t_(3 mesi) = 94.2%;t_(6 mesi) = 94.3% Moisture content   ≦10% Resistence to crushing ≧20NHPLC Title T3S 90-110% HPLC Title T4 90-110%

Stability tests were carried out demonstrating that tablets are stablefor at least 24 months.

Example D Quantitation of T₃S by Immunoassay with ChemiluminescenceDetection

Synthesis of T₃S Biotin Derivative

Briefly, T₃S biotin derivative was synthesized as follows:N-hydroxysuccinimidyl d-biotin-15-amido-4,7,10,13-tetraoxapentadecylateA (50 mg; 0.0849 mmol) was solubilized in DMAC (2 mL), to which DIPEA(14.5 uL; 0.0866 mmol) was added, while maintaining the reaction mixtureunder continuous stiffing at 0° C. T₃S (68.4 mg; 0.0908 mmol, preparedas described in Mol & Visser, Endocrinology 1985, 117:1-7) was thenadded and after a few minutes the suspension was left to heat up to roomtemperature to give a clear solution. It was allowed to stir for 2 h,then kept overnight at the same temperature. DMAC was evaporated underreduced pressure (10 mbar; 40° C.) to give a colourless oil. The crudeso obtained was dissolved in H₂O and purified by Semi-preparative HPLC.The fractions containing the product were collected, concentrated andfinally lyophilized to give T₃S-biotin as a white solid (59.6 mg; 0.0495mmol). Yield 58%.

A polyclonal anti-T₃S antiserum was obtained in rabbits as described inChopra et al., J. Clin. Endocrinol. Metab., 1992, 75: 189-194.

The assay was based on a competitive ELISA in which increasing amountsof T₃S competed for the antibody binding with a fixed amount of T₃Sconjugated with biotin, in a white 96 well plate. The employment of thebiotin-avidin interaction, which allows signal amplification, combinedwith luminescence as technique for signal development allowed for asensibility comparable to the RIA test (described in Chopra et al., J.Clin. Endocrinol. Metab., 1992, 75: 189-194).

Standard solutions of T₃S were prepared at the following concentrations:1000, 200, 40, 8, 1.6 pg/mL in Diluent Buffer: PBS, 0.05% Tween, 0.3%BSA

The tracer solution (T₃S-Biotin, 180.6 μM) was prepared in the abovediluent buffer. Antibody solution: T₃S rabbit antiserum was diluted1:50000 in Diluent Buffer plus 8 mM ANS, 1.2 mg/mL Sodium Salicylate.

A 96 well white plate was coated over night at 4° C. with 100 μL/well of2 μg/mL anti Rabbit IgG diluted in phosphate buffer pH 7.8. At the sametime, Standard solutions of biotin labelled T₃S were combined with thediluted antiserum and the T₃S-biotin solution as reported in Table A.The mixed samples were incubated at room temperature in the dark, overnight.

The day after, the plate was washed four times with Washing Buffer(0.05% Tween 20 in PBS), then incubated in Blocking Buffer (2% BSA inWashing Buffer) for 1 h at room temperature.

Afterwards, the plate was rinsed four times with Washing Buffer, 100μL/well of the mixed samples were added in triplicate and the plate wasincubated 3 h at room temperature.

Then, the plate was rinsed three times with Washing Buffer and incubatedwith Streptavidin Poly-HRP (10 ng/mL in RASA, 100 μL/well) for 1 h atroom temperature. After additional six washes, the plate was incubatedwith SuperSignal ELISA Femto Maximum Sensitivity Substrate (100 μL/well)for 5 min in the dark and the emitted light was read as counts persecond (CPS) with a luminescence plate reader

TABLE A Calibration Curve Preparation T3S/1 T3S/1 Antiserum T3S-biotin(μL) (μL) (μL) CS 5 (1000 pg/mL) 250 125 50 CS 4 (200 pg/mL) 250 125 50CS 3 (40 pg/mL) 250 125 50 CS 2 (8 pg/mL) 250 125 50 CS 1 (1.6 pg/mL)250 125 50 B0 — 125 50 NSB — — 50

The calibration curve was prepared in buffer using five concentrationsof the test item in the range 1.6-1000 pg/mL. The curve is shown in FIG.1, panel a).

Example E Quantitation of T₃S by DELFIA®

Preparation of:

[[3,5-Diiodo-N-[[(carboxymethyl)[2-[(carboxymethyl)[2-[bis(carboxymethyl)amino]ethyl]amino]ethyl]amino]acetyl]-O-[3-iodo-4-(sulfooxy)phenyl]-L-tyrosinate(6-)]europate(3-)]trisodium(Formula II).

Synthesis of Eu-DTPA-T₃S Monoamide

The reaction scheme of the synthesis of3,5-Diiodo-N-[[(carboxymethyl)[2-[(carboxymethyl)[2-[bis(carboxymethyl)amino]ethyl]amino]ethyl]amino]acetyl]-O-[3-iodo-4-(sulfooxy)phenyl]-L-tyrosine(DTPA-T₃S monoamide) is shown in FIG. 2.

A solution of H₂O (0.282 ml; 15.64 mmol) in DMAC (43 mL) was addeddropwise to a suspension of N,N-bis[2-(2,6-dioxylenolorange-4-morpholinyl)ethyl]glycine A (4.27 g; 11.94 mmol) in DMAC (85mL) at room temperature. At the end of the addition the mixture washeated to 80° C. After 4.5 h the reaction mixture was cooled to 25° C.and a solution of T₃S/1 (3 g; 3.98 mmol) and DIPEA (2.71 mL; 15.92 mmol)in DMAC (85 mL) was added dropwise over 20 min DMAC was evaporated underreduced pressure (10 mbar; 40° C.). The oily residue was diluted withH₂O (200 mL), obtaining precipitation of a yellowish solid that wasfiltered washed with H₂O and dried. The crude so obtained was dissolvedin Acetone/H₂O 20:80 (v/v), the solution (pH=2,97) was loaded on anAmberlite® XAD-1600 resin column (200 mL; diam. 6 cm) and eluted with aAcetone/H₂O gradient. The fractions containing the product havingsimilar composition were collected and evaporated to give the ligandDTPA-T₃S as a solid (1.27 g; 1.15 mmol). Yield 26%.

Europium chloride hexahydrate (0.17 g, 0.46 mmol) was added in portionsto a solution of the ligand DTPA-T₃S (0.51 g; 0.46 mmol) in H₂O (50 mL)at 20° C. (pH 2.93); after each addition the suspension was stirreduntil complete dissolution. Once the complexation was complete the pHwas adjusted to 7 with 0.1 N NaOH and the solution was desalted byelution with water/acetone from a column of Amberlite® XAD-1600 resin(100 mL; diam. 3 cm). The fractions containing the desired product andfree from salts were collected and evaporated to give the compound ofFormula I (0.37 g, 0.28 mmol) a yellow solid. Yield: 61%.

The immunoassay method and solutions were as described in the Example D,with the following exceptions: a DELFIA® Wash (Perkin Elmer) was usedinstead of the above Washing buffer. The Tracer stock solution containedthe Europium 100 μM and it was stored at +4° C., protected from light.Just before use it was diluted 1:300000 in Assay Buffer to obtain afinal concentration of 440 pg/mL.

The assay was performed in Delfia Yellow plates (Perkin Elmer).

After the 3-h incubation with the mixed samples, the Formula II dilutedcompound solution was added (50 μL per well) to all wells. The plateswere then sealed with plastic adhesive sheets and incubated underagitation for 1 h at 37° C.

After three washes, the plates were tapped dry on absorbent paper, andDelfia Enhancement Solution (Perkin Elmer) was added (200 μL) After 1 hat 25° C., the plates were read in a Victor3 instrument according to the“Europium” manufacturer protocol.

A calibration curve was prepared using nine concentrations of the testitem in the range 30-2000 pg/mL. The curve is shown in FIG. 1, panel b).

Example F Clinical Trial

1) Ethical Issues

This study was conducted in Pisa, Italy under the guidelines provided inthe Declaration of Helsinki, ICH E6 Guideline for Good ClinicalPractice, and the requirements of the European Directive 2001/20/EC, andLaw Decree Jun. 24, 2003, n. 211 implementing Directive 2001/20/CE inItaly, as well as the European Commission Directive 2005/28/EC of 8 Apr.2005, laying down principles and detailed guidelines for good clinicalpractice for investigational medicinal products for human use, as wellas the requirements for authorisation of the manufacturing orimportation of such products, and related guidance.

2) Safety

The study was designed to guarantee that plasma levels of total T₃ couldnot exceed 196.6 ng/dl, the level obtained by the administration of theconsolidated standard therapy of 20 μg T₃.

3) Protocol

About 30 human subjects with surgically excised thyroids wereadministered a to single dose of an oral T₃S composition of theinvention containing 20, 40, 80 or 160 μg T₃S in tablet form. Serumlevels of thyroid hormone including T₃S and triiodothyronine (“T₃”) asboth free T₃ (“FT₃”) and total T₃ (“TT₃”) were assessed by T₃S RIA, asdescribed in Chopra et al., J. Clin. Endocrinol. Metab., 1992, 75:189-194.

Forty eight hours prior to administration of the oral T₃S composition ofthe invention, patients were screened for the study criteria andinformed consent was requested and obtained. Twenty-four hours prior toadministration of the oral T₃S composition of the invention the subjectwas examined and all specimens for laboratory tests were collected,including thyroid function tests. On the day of the administration ofthe oral T₃S composition of the invention, a further check of theinclusion/exclusion criteria was performed and patients were given asingle dose of the oral T₃S composition of the invention in tablet formaccording to the dose group in which they were placed.

The tablet composition was as follows:

Ingredient Amount per Tablet T₃S sodium salt 20.6 μg Equivalent to T₃S20 μg Calcium carbonate 30 mg Glycerol dibehenate 5 mg Croscarmellosesodium salt 3.5 mg Hydrate colloidal silica 2 mg Magnesium stearate 0.5mg Microcrystalline cellulose To 110 mg

The initial part of the study was aimed at determining the optimal dose:as none of the patients treated with the 20, 40, 80 and 160 μg doses ofthe oral T₃S composition of the invention had serum levels of TT₃exceeding 196.6 ng/dl, the 160 μg dose was selected for use in thesecond part of the study.

12 subjects received a single dose of the oral composition of theinvention containing 160 μg T₃S. The absorption of T₃S was assessed bymeasuring the serum levels of thyroid hormones TT₃, FT₃, T₃S, freethyroxine (“FT₄”) and Thyrotropin (or Thyroid Stimulating hormone,“TSH”).

T₃S in serum was detected with a peak level two hours afteradministration of the oral composition, as shown in FIG. 1. In patientslacking a thyroid there is no endogenous T₃. Thus, all T₃ present in thesubjects was the result of conversion of T₃S from the oral compositionsto T₃ in vivo. By monitoring serum T₃S and TT₃ levels afteradministration of the oral T₃S compositions, it was determined that T₃Swas converted to the clinically active TT₃ in a dose related fashion.

Serum levels of TSH and FT₄ were determined at 24 h and 30 minutes priorto administration, and at 24 and 48 hours±15 min after theadministration of T₃S composition. Gastrointestinal absorption of T₃Swas assessed by measurement of circulating serum concentrations of TT₃,T₃S and FT₃. Circulating serum concentrations of FT₃ was measured preand post dose to verify the in-vivo T₃S-FT₃ conversion in patients.

Safety and tolerability were assessed by monitoring adverse events andby monitoring effects on vital signs, ECG, hematology, blood chemistryand urinalysis after administration of the oral compositions of theinvention.

4) Conclusions

Regardless of dose, the oral T₃S compositions of the invention werefound to be safe and well tolerated. The mean serum concentration of T₃S(in ng/dl) for each of the four dose groups in the initial part of thestudy is shown in FIG. 3. For each dose group T₃S was present in theserum, with a peak level two hours after oral administration. As allsubjects were thyroidectomised and thus lacking endogenous T₃S this dataestablishes that the T₃S from the oral compositions of the inventioncrosses the gastrointestinal tract and enters the bloodstrem.

The mean serum concentration of T₃S and TT₃ after administration of a160 μg dose of the oral composition of the invention is shown in FIG. 4for a patient. TT₃ was detected within 4-5 hours of administration ofthe oral T₃S. As all subjects were thyroidectomised and thus lackingendogenous T₃, the only T₃ source is exogenously administered T₃S.

This data establishes that T₃S is absorbed (i.e. it crosses theGastrointestinal Barrier) and is found in serum after oraladministration, is converted to the clinically active T₃ in adose-related fashion and that T₃ levels in serum are still detectable 48hrs after single dose administration.

REFERENCES

-   1. Chopra I J. Nature, source and relative biological significance    of circulating thyroid hormones. In: Braverman L E., Utiger R D.    (eds) The Thyroid, Lippincott, Philadelphia 1991, pp. 126-143.-   2. Spaulding S W., Smith T J., Hinkle P M., Davis F B., Kung M P.,    Roth J A. Studies on the biological activity of triiodothyronine    sulfate. J. Clin. Endocrinol. Metab. 1992, 74, 1062-1067.-   3. Lo Presti J S., Mizuno L., Nimalysuria A., Anderson K P., Spencer    C A., Nicoloff J T. Characteristics of 3,5,3′-triiodothyronine    sulfate metabolism in euthyroid man. J. Clin. Endocrinol. Metab.    1991, 73, 703-709.-   4. Santini F., Hurd R E., Chopra I J. A study of metabolism of    deaminated and sulfoconjugated iodothyronines by rat placental    iodothyronine 5-monodeiodinase. Endocrinology 1992, 131, No. 4,    1689-1694.-   5. Otten M H., Mol J A., Visser T J. Sulfation proceeding    deiodination of iodothyronines in rat hepatocytes. Science 1983,    221, 81-83.-   6. Mol J A., Visser T J. Rapid and selective inner ring deiodination    of T₄ sulfate by rat liver deiodinase. Endocrinology 1986, 117,    8-12.-   7. Kung M P., Spaulding S W., Roth J A. Desulfation of    3,5,3′-triiodothyronine sulfate by microsomes from human and rat    tissues. Endocrinology 1988, 122, 1195-1200.-   8. Santini F., Chopra I J., Wu S Y., Solomon D H., Chua Teco G N.    Metabolism of 3,5,3′-triiodothyronine sulfate by tissues of the    fetal rat: a consideration of the role of desulfation of    3,5,3′-triiodothyronine sulfate as a source of T₃. Pediatr. Res.    1992, 31, 541-544.-   9. De Herder W W., Hazenberg M P., Pennock-Schroeder A M., Hennemann    G., Visser T J. Rapid bacteria-dependent in vitro hydrolysis of    iodothyronine conjugates by intestinal contents of humans and rats.    Med. Biol. 1986, 64, 31-35.-   10. Santini F., Hurd R E., Lee B., Chopra I J. Thyromimetic effects    of 3,5,3′-triiodothyronine sulfate in hypothyroid rats.    Endocrinology 1993, 133, No. 1, 105-110.-   11. Santini F., Chiovato L., Ghiri P., Lapi P., Mammoli C.,    Montanelli L., Scartabelli G., Ceccarini G., Coccoli L., Chopra I    J., Boldrini A., Pinchera A. Serum iodothyronines in human fetus and    the newborn: evidence for an important role of placenta in fetal    thyroid hormone homeostasis. J. Cl. Endocrinol. Metab. 1999, 84, No.    2, 493-498.-   12. Santini F., Pinchera A., Ceccarini G., Castagna M., Rosellini    V., Mammoli C., Montanelli L., Zucchi V., Chopra I J., Chiovato L.    Evidence for the role of the type III-iodothyronine deiodinase in    the regulation of 3,5,3′-triiodothyronine content in the human    central nervous system. Eur. J. Endocrinol. 2001, 144, 577-583.-   13. Santini F., Cortellazzi D., Baggiani A M., Marconi A M.,    Beck-Peccoz P., Chopra I J. A study of the serum    3,5,3′-triiodothyronine sulfate concentration in to normal and    hypothyroid fetuses at various gestational stages. J. Cl.    Endocrinol. Metab. 1993, 76, No. 6, 1583-1587.-   14. Chopra I J., Nguyen D. Demonstration of thyromimetic effects of    3,5,3′-triiodothyronine sulfate (T₃S) in Euthyroid rats. Thyroid    1996, 6, No. 3, 229-232.    Embodiments of the Invention-   1. Triiodothyronine sulfate for use as a medicament.-   2. Triiodothyronine sulphate for use as a medicament according to    embodiment 1, having thyromimetic activity.-   3. Triiodothyronine sulfate according to embodiment 2, for use in    the treatment of pathologies due to organic deficiency of    triiodothyronine.-   4. Triiodothyronine sulfate according to embodiment 3, wherein said    pathologies comprise original hypothyroidism from autoimmune thyroid    affections, hormonal production defects, thyroidectomy, congenital    hypothyroidism.-   5. Triiodothyronine sulfate according to embodiment 2, for use in    the treatment of disorders due to reduced activity of type I    5′-iodothyronine monodeiodinase.-   6. Triiodothyronine sulfate according to embodiment 5, wherein said    reduced activity of type I 5′-iodothyronine monodeiodinase    comprises, among its grounds, hypothyroidism, non thyroidal systemic    illnesses, fast, selenium shortage.-   7. Pharmaceutical compositions comprising triiodothyronine sulfate    as an active principle.-   8. Pharmaceutical compositions according to embodiment 7, wherein    said triiodothyronine sulfate is formulated in association with    thyroxine.-   9. Pharmaceutical compositions according to embodiment 7 and 8,    wherein said compositions further comprise additives like    excipients, diluents, dissolvents, solvents, carriers, dyestuffs,    flavourings, sweeteners.-   10. Pharmaceutical compositions according to embodiment 7, wherein    triiodothyronine sulfate is administered at doses raging from 5 to    1000 μg.-   11. Pharmaceutical compositions according to embodiment 10, wherein    triiodothyronine sulfate is administered at doses raging from 10 to    500 μg.-   12. Pharmaceutical compositions according to embodiment 10, wherein    triiodothyronine sulfate is administered at doses raging from 25 to    250 μg.-   13. Pharmaceutical compositions according to embodiment 8, wherein    said association is administered at doses ranging from 10 to 500 μg    of triiodothyronine sulfate and from 10 to 250 μg of thyroxine.-   14. Pharmaceutical compositions according to embodiment 8, wherein    said association is administered at doses ranging from 25 to 250 μg    of triiodothyronine sulfate and from 25 to 200 μg of thyroxine.-   15. Kit for the differential or sequential administration of the    pharmaceutical compositions according to embodiments 8, 9 and 11 to    14.-   16. Use of triiodothyronine sulfate for the preparation of the    pharmaceutical compositions according to embodiments 7 to 15.

What is claimed is:
 1. A diagnostic non-radioactive immunoassay for T₃Squantitation in serum comprising a anti-T₃S antibody and anon-radioactive T₃S-conjugate comprising either an avidin derivative ora lanthanide chelating moiety.
 2. The immunoassay of claim 1 wherein thenon-radioactive conjugate is T₃S-biotin.
 3. The immunoassay of claim 2wherein the assay is a competitive ELISA.
 4. The immunoassay of claim 1wherein the non-radioactive T₃S conjugate comprises the Lanthanidechelating agent of Formula I


5. A kit comprising a first container for a non-radioactive immunoassayaccording to claim 1 and a second container for a solid oral dosagecomposition comprising T₃S as the active principle, in a quantityranging from 1 to 1000 μg and further comprising diluents anddisintegrants and also glidants and/or lubricants.
 6. The kit of claim5, wherein the solid oral dosage of the second container furthercomprises excipients, stabilizers, preservatives or dissolutionenhancers.